Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Nitrogen containing other than solely as a nitrogen in an...
Reexamination Certificate
2001-03-28
2003-05-27
Davis, Brian (Department: 1621)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Nitrogen containing other than solely as a nitrogen in an...
C514S640000, C514S645000, C564S248000, C564S300000
Reexamination Certificate
active
06569902
ABSTRACT:
BACKGROUND
Chronic feeding of a choline-deficient-L-amino acid-defined (CDAA) diet containing no carcinogens exerts a strong hepatocarcinogenicity in rats through the development of apparently preneoplastic, focal lesions in the background presence of repeating hepatocyte death and regeneration as well as fibrosis. Oxidative stress appears to play major roles in its underlying mechanisms in association with alteration on the status of various signaling molecules. Phenyl N-tert-butyl nitrone (PBN), a radical trapper, has been shown to inhibit the development of preneoplastic lesions in the early phase of this dietary hepatocarcinogenesis by apparently inhibiting oxidative stress, inducible cyclo-oxygenase activity and fibrogenesis (Floyd et al., 1998).
Reactive oxygen species (ROS) have been implicated in cancer development for many years. A prime example where ROS are strongly implicated is the model system where feeding a choline deficiency (CD) diet to rats leads to hepatocellular carcinoma (HCC) development, i.e. in the complete absence of exposure to any exogenous known carcinogen. Utilizing this model, the present invention concerns novel observations that make it possible to link ROS with key signal transduction pathways that have been shown to be fundamental in cancer initiation and development. The present inventors have shown that mitochondria from CD-livers are changed such that they mediate a significantly higher yield of H
2
O
2
production. Additionally, for the first time the present inventors have shown that PBN (&agr;-phenyl-tert-butyl nitrone) and its derivatives are nitrone-based free radical traps and, significantly reduce preneoplastic nodule development as well as inhibit hepatocellular carcinoma (HCC) formation at very low levels of the compound. PBN and the like are the most potent anti-carcinogens ever studied in this model. To understand these observations the inventors postulate that the CD-regimen mediates changes in mitochondrial membranes such that they produce enhanced levels of H
2
O
2
and that PBN and the like significantly inhibit the excess H
2
O
2
production by acting at Complex I. The present inventors further postulate that excess H
2
O
2
causes an enhanced inactivation of the PTEN tumor suppressor protein, which causes a loss of its phosphatase activity and thereby mediates a shift toward the activation of the AKT-kinase pathway resulting in a decrease in apoptosis-mediated processes but an increase in oncogenic events. The inventors also propose that the cells in preneoplastic nodules which develop in CD-livers are predisposed toward ontogenesis (as opposed to apoptosis) because of the action of excess H
2
O
2
and certain growth factors (most likely TGF&bgr;
1
) and that PBN and the like alter these processes through both inhibition of excess H
2
O
2
production and also by suppression of enhanced signal transduction processes. The inventors believe that PBN and the like act to cause preneoplastic nodule cells to become predisposed toward apoptic processes leading to inhibition of tumor development.
Studies on the Pharmacological Action of PBN
The compound PBN was first synthesized in the 1950's, but in 1968 it was discovered to be very useful to trap and stabilize free radicals in chemical reactions and hence it was termed a spin-trap (Janzen 1971). Although PBN is the prototype spin-trap several other nitrones have been synthesized and found useful to trap and characterize free radicals in chemical reactions. These spin traps were used in chemical reactions first, but in the mid-1970's they began to be used to trap free radicals in biochemical and biological systems (Floyd et al. 1978; and Poyer et al. 1978, for example). Pharmacokinetic studies have shown that PBN is readily and rapidly distributed almost equally to all tissues, has a half-life in rats of about 132 minutes and is eliminated mostly in the urine. Relatively few metabolism studies have been done, but it is known that some ring hydroxylation (primarily in the para position) of the compound occurs in the liver. Novelli first showed that PBN could be used to protect experimental animals from septic shock (Novelli et al. 1986), and indeed this was later confirmed by other groups (Pogrebniak et al. 1992). The use of PBN and derivations as pharmacological agents began after discoveries in 1988 that showed that PBN had neuroprotective activity in experimental brain stroke models (Floyd 1990; Floyd et al. 1996; and Carney et al. 1991). These results were repeated and extended, (i.e. see References Clough et al. 1991; Cao et al. 1994; Folbergrovaet al. 1995; Pahlmark et al. 1996, for example). The present inventors have summarized the extensive neuroprotective pharmacological research effort on PBN and derivatives (Floyd 1997; Hensley et al. 1996). In addition to neurodegenerative diseases, PBN has been shown to protect in other pathological conditions where ROS-mediated processes are involved, including diabetes and many other conditions. The mechanistic basis of why PBN and some of its derivatives are so neuroprotective in experimental stroke and several other neurodegenerative models has not been completely elucidated yet. However, it is clear that its action cannot simply be explained by its ability to trap free radicals. In fact the present inventors' research effort on the mechanistic basis of PBN's action now shows that it is acting by suppressing gene induction (Floyd 1997; Hensley et al. 1996; Miyajima et al. 1995; Tabatabaie et al. 1996; and Hensley et al. 1997), most likely by acting on oxidation-sensitive signal transduction processes (Robinson et al. 1999). In fact PBN seems to be acting by suppressing signal transduction enhanced ROS formation by mitochondria (Hensley et al. 1998). These findings and ideas have arisen from the study of neurodegenerative processes. It should be emphasized, however, that PBNs action in preventing CD carcinogenesis may be different than those found in the neurodegenerative disease models. A specific mechanism of action does not limit the present invention.
PBN is Protective in Choline-deficiency Model
Earlier studies showed that PBN administered in drinking water was very protective in the CD-model. The results were assessed after 12 weeks on the regime (Nakae et al. 1998). The research brought out several important points (1) PBN, even at the lowest level, drastically reduced the size of neoplastic nodules (from 1.92 mm
3
in CDAA only to 0.33, 0.17 and 0.10 mm
3
for the CDAA plus PBN treated at 6, 30 and 60 mg/kg-day respectively, see Table 1 of Nakae et al. 1998).
There was less effect of PBN on nodule number, i.e. 190 per mm
3
for CDAA only to 170, 149 and 142 for the 6, 30 and 60 mg/kg- day respectively, (see Table 1 of Nakae et al. 1998). (2) PBN significantly reduced connective tissue proliferation. (3) Increasing concentrations of PBN reduced 8-OHdG content (a marker of DNA oxidation) in the CD-livers. (4) PBN reduced the amount of PGE
2
in the CD-livers by about 50% at the highest dose but it had no effect on COX-II expression, either the mRNA or protein level. In summary then the fact that the very lowest level of PBN decreased the nodule size by 83% but only decreased the nodule number by 11% indicates to us that nodule size is the most sensitive parameter to PBN treatment. There was some effect on PGE
2
levels but only at the highest levels of PBN and this probably had to do with it acting as a catalytic inhibitor of the enzyme per se.
To highlight the potency of PBN relative to other chemicals that have been tested in the CDAA model, it is instructive to compare results, which were obtained by the Nakae-Konishi group (see Mizumoto et al. 1994; Endoh et al 1996; and Nakae 1999). The data clearly show that PBN is the most effective compound tested in the CDAA regimen in reducing the size of the preneoplastic nodules and in preventing an increase in the 8-OHdG content. The effectiveness of PBN on nodule size is much more potent than comparable amounts of the other inhibitors, most of which are free
Floyd Robert A.
Hensley Kenneth L.
Kotake Yashige
Nakae Dai
Davis Brian
Fulbright & Jaworski
Oklahoma Medical Research Foundation
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